The long-term goal of this research is to develop improved agents or strategies for the treatment of human cancer by exploiting aspects of folyl or antifolylpolyglutamate synthesis. Folypolyglutamates are essential for viability and proliferation; polyglutamates of classical antifolates are implicated in the cytotoxic mechanism of these agents. Synthesis of polyglutamates plays a role in drug resistance and may also have a role in selectivity. Complete understanding of the synthesis and function of folyl- and antifolylpolyglutamates may expedite the development of new agents to exploit this critical pathway. This goal will be addressed through the following specific aims: (1) design and characterization of inhibitors of folypolyglutamate synthetase (FPGS), the enzyme which synthesizes polyglutamates. Rational design of these agents is based on FPGS substrate specificity and inhibitor data generated in this lab. Syntheses will be performed by collaborators. Characterization of the agents will be performed in this lab. Several classes of FPGS inhibitors are proposed, including (a) analogs of the only known, human FPGS- specific inhibitor, 5,8-dideazapteroylornithine (Dr. Piper) and (b) the gamma-tetrazole folate analogs (Dr. Kalman) which seem to be well transported; (2) use of FPGS inhibitors in the investigation of polyglutamate metabolism and regulation in whole cells. Basic knowledge derived from such studies may allow the more effective use of current antifols or suggest new targets for therapeutic intervention; (3) characterize existing human leukemia cell sub- lines which are resistant to MTX solely because of decreased MTX polyglutamate (MTXGn) synthesis. These lines are unique both in their clinically relevant selection method and the fact that they apparently have no other mechanisms of resistance. One of these lines contains an altered FPGS; this is the only description of MTX resistance due to an altered FPGS. Since decreased MTXGn synthesis has been identified clinically in acquired resistance to MTX, this cell line presents an opportunity to characterize this phenotype and to design therapeutic strategies to overcome this type of resistance. Comparison of the enzymatic properties of the native and altered FPGS will also provide information which will aid design of FPGS inhibitors.